IL-23 is implicated in the pathogenesis of immune-mediated inflammatory diseases, and myeloid cells that express Fc gamma receptor 1 (Fc gamma RI or CD64) on their surface have been recently identified as a primary source of IL-23 in inflamed tissue. Our complementary analyses of transcriptomic datasets from psoriasis and IBD showed increased expression of CD64 and IL-23 transcripts in inflamed tissue, and greater abundance of cell types with co-expression of CD64 and IL-23. These findings led us to explore potential implications of CD64 binding on the function of IL-23-targeting monoclonal antibodies (mAbs). Guselkumab and risankizumab are mAbs that target the IL-23p19 subunit. Guselkumab has a native Fc domain while risankizumab contains mutations that diminish binding to Fc gamma Rs. In flow cytometry assays, guselkumab, but not risankizumab, showed Fc-mediated binding to CD64 on IFN gamma-primed monocytes. Guselkumab bound CD64 on IL-23-producing inflammatory monocytes and simultaneously captured IL-23 secreted from these cells. Guselkumab binding to CD64 did not induce cytokine production. In live-cell confocal imaging of CD64+ macrophages, guselkumab, but not risankizumab, mediated IL-23 internalization to low-pH intracellular compartments. Guselkumab and risankizumab demonstrated similar potency for inhibition of IL-23 signaling in cellular assays with exogenous addition of IL-23. However, in a co-culture of IL-23-producing CD64+ THP-1 cells with an IL-23-responsive reporter cell line, guselkumab demonstrated Fc-dependent enhanced potency compared to risankizumab for inhibiting IL-23 signaling. These in vitro data highlight the potential for guselkumab binding to CD64 in inflamed tissue to contribute to the potent neutralization of IL-23 at its cellular source.

IL-23 is implicated in the pathogenesis of immune-mediated inflammatory diseases, and myeloid cells that express Fc gamma receptor 1 (FcγRI or CD64) on their surface have been recently identified as a primary source of IL-23 in inflamed tissue. Our complementary analyses of transcriptomic datasets from psoriasis and IBD showed increased expression of CD64 and IL-23 transcripts in inflamed tissue, and greater abundance of cell types with co-expression of CD64 and IL-23. These findings led us to explore potential implications of CD64 binding on the function of IL-23–targeting monoclonal antibodies (mAbs). Guselkumab and risankizumab are mAbs that target the IL-23p19 subunit. Guselkumab has a native Fc domain while risankizumab contains mutations that diminish binding to FcγRs. In flow cytometry assays, guselkumab, but not risankizumab, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. Guselkumab bound CD64 on IL-23–producing inflammatory monocytes and simultaneously captured IL-23 secreted from these cells. Guselkumab binding to CD64 did not induce cytokine production. In live-cell confocal imaging of CD64+ macrophages, guselkumab, but not risankizumab, mediated IL-23 internalization to low-pH intracellular compartments. Guselkumab and risankizumab demonstrated similar potency for inhibition of IL-23 signaling in cellular assays with exogenous addition of IL-23. However, in a co-culture of IL-23–producing CD64+ THP-1 cells with an IL-23–responsive reporter cell line, guselkumab demonstrated Fc-dependent enhanced potency compared to risankizumab for inhibiting IL-23 signaling. These in vitro data highlight the potential for guselkumab binding to CD64 in inflamed tissue to contribute to the potent neutralization of IL-23 at its cellular source.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Abstract Background IL-23 is implicated in the pathogenesis of inflammatory bowel disease (IBD) and myeloid cells that express FcγRI (CD64) have been identified as the primary cellular source of IL-23 in inflamed IBD gut tissue. Guselkumab (GUS) and risankizumab (RZB) are monoclonal antibodies (mAbs) specifically directed against the IL-23p19 subunit. GUS is a fully human IgG1 mAb with a native Fc region while RZB is a humanized IgG1 mAb with a mutated Fc region. Here, we evaluated CD64 and IL-23 expression in IBD patient gut biopsies, binding of GUS and RZB to CD64, and the functional consequences of CD64 binding by IL-23p19 subunit mAbs, in in vitro assays. Methods IL23A and FCGR1A (CD64) expression was analyzed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes, as well as binding to IL-23–secreting inflammatory monocytes and capture of endogenously secreted IL-23, were assessed by flow cytometry. Internalization of IL-23, GUS, and RZB within CD64+ macrophages was evaluated using live cell confocal imaging. Potency of GUS and RZB for inhibiting IL-23 signaling was determined in a co-culture of THP-1 (a CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). Results Analysis of RNAseq datasets showed that FCGR1A, IL23A, and IL12B were significantly increased in inflamed versus non-inflamed IBD gut biopsies and that IL23A was predominantly expressed by FCGR1A-expressing myeloid cells. In in vitro assays GUS, but not RZB, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS, but not RZB, bound to the surface of CD64+ macrophages and mediated internalization of IL-23 to low pH intracellular compartments. GUS and RZB demonstrated similar potency for inhibiting signaling by IL-23 present in THP-1–conditioned media. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency compared to RZB for inhibition of IL-23 signaling. Conclusion Our transcriptomic analysis supported previous observations of CD64+ myeloid cells as a key source of IL-23 production in inflamed IBD gut tissue. GUS binding to CD64 on IL-23–producing cells likely contributed to the enhanced functional potency of GUS compared to RZB for inhibition of IL-23 signaling in the co-culture assay. These in vitro data support a hypothesis for optimal enrichment of GUS in inflamed tissues where CD64+ IL-23–producing myeloid cells are increased and in proximity to IL-23–responsive lymphoid cells, enabling GUS to more potently neutralize IL-23 by targeting IL-23 at its source of production.

Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory disease characterized by complex lesion morphology including nodules, abscesses, and epithelialized sinus tracts.The heterogeneity of HS histopathology has not been fully elucidated and represents a challenge in comprehensive molecular analyses.Current studies have categorized transcriptomes of lesional and perilesional tissue, but none have analyzed HS tunnels.To overcome the complexity of HS histological features and obtain the gene expression of defined histopathological structures, we performed retrospective analysis of FFPE tissue specimens.FFPE samples from HS lesional, tunnel, and perilesional tissue (n¼5 each) and age, gender, and location-matched healthy skin controls (n¼6) were used for RNA and DNA isolation and subsequent qPCR of pro-inflammatory mediators coupled with the 16s rRNA based microbiota quantification.Immunohistochemical staining for myeloperoxidase and CD45 was utilized to confirm the number of infiltrates, while immunofluorescence staining for keratin 17 (K17) evaluated activated keratinocytes.Simultaneous extraction of RNA and DNA was optimized from a FFPE tissue determining 4 mm biopsy as sufficient.Real-time qPCR data confirmed consistent upregulation of IFN-g, IL-6, and IL-8 in all HS samples and variability in TNF-a, IL-1b, and IL-23 expression corresponding to the amount of immune cells in the FFPE tissue.Furthermore, location-specific K17 staining was predominantly associated with the HS tunnels.DNA isolated from the same samples was successfully utilized for 16s rRNA qPCRbased bacterial quantification.This method allows for comprehensive evaluation of HS pathology from a defined FFPE sample enabling simultaneous host-microbiome evaluation from a single 4 mm biopsy.Our data also provide a rationale for the utilization of this method to guide clinical trials and outcomes allowing further understanding disease progression.

Background Monoclonal antibodies targeting the interleukin (IL)-23p19 subunit are effective in the treatment of psoriatic disease but have different molecular attributes that may translate to differences in clinical efficacy. Within this class, guselkumab (GUS) is a fully human IgG1 monoclonal antibody with a native Fc region, while risankizumab (RIS) is a humanised IgG1 antibody with a mutated Fc region. Binding of these therapeutic antibodies to Fcγ receptor (FcγR) I, also known as CD64, is of interest, as CD64+ IL-23-producing myeloid cells are increased within inflamed tissue of patients with psoriatic disease [1]. Furthermore, the incidence and prevalence of psoriatic arthritis increases with the severity of psoriasis [2], and joint disease activity is positively correlated with frequency of peripheral CD64+ monocytes [3]. Objectives Functional characteristics of the antigen-binding and Fc regions of GUS and RIS were compared. Methods IL-23 binding affinity was evaluated in vitro using a kinetic exclusion assay (KinExA) and surface plasmon resonance. In vitro cellular potency was measured by impact on IL-23-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human peripheral blood mononuclear cells. Binding of GUS and RIS to FcγRs was assessed in cells transfected with individual FcγRs. Primary human “inflammatory” monocytes differentiated with granulocyte-macrophage colony-stimulating factor and interferon-γ (IFN-γ) were induced to produce IL-23 via toll-like receptor stimulation and used to assess binding of GUS and RIS to CD64 and potential capture of endogenously secreted IL-23 by flow cytometry. The potential for GUS binding to CD64 on IFN-γ primed monocytes to trigger activation was assessed using a 41-plex cytokine bead assay. Results GUS and RIS displayed comparable picomolar binding affinity for IL-23 and equivalent high potency for inhibiting IL-23-induced STAT3 phosphorylation. GUS showed strongest binding to CD64 compared with other FcγRs, whereas RIS had negligible binding to any FcγR. GUS, but not RIS, showed dose-dependent Fc-mediated binding to CD64 on primary human “inflammatory” monocytes. Moreover, CD64-bound GUS was able to simultaneously capture IL-23 endogenously secreted from the same cells (Figure 1). GUS binding to CD64 on monocytes did not induce cytokine production. Conclusion GUS, but not RIS, simultaneously binds CD64+ myeloid cells via its Fc region and neutralises IL-23 with high affinity and potency. Our in vitro data suggest a mechanistic benefit through enrichment of GUS within inflamed tissue of patients with psoriatic disease, where CD64+ IL-23-producing myeloid cells are increased, such that GUS potently neutralises IL-23 at its source of production. These findings may contribute to differences in clinical-therapeutic profiles between antibodies. References [1]Mehta, H. et al. J Invest Dermatol. 2021;141:1707-1718.[2]Merola, J. et al. J Am Acad Dermatol. 2022;86:748-757.[3]Matt, P. et al. Scand J Rheumatol. 2015;44:464-73. Acknowledgements: NIL. Disclosure of Interests Dennis McGonagle Consultant of: AbbVie, Amgen, Bristol Myers Squibb, Celgene, Eli Lilly, Gilead, Janssen, Novartis, Pfizer, and UCB, Grant/research support from: AbbVie, Amgen, Bristol Myers Squibb, Celgene, Eli Lilly, Gilead, Janssen, Novartis, Pfizer, and UCB, Raja Atreya Consultant of: AbbVie, Amgen, Arena Pharmaceuticals, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion Healthcare, Dr. Falk Pharma, Ferring, Fresenius Kabi, Galapagos, Gilead, GlaxoSmithKline, InDex Pharmaceuticals, Janssen, Kliniksa Pharmaceuticals, Merk Sharp & Dohme, Novartis, Pfizer, Roche, Samsung Bioepsis, Stelic, Sterna Biologicals, Takeda, and Tillotts, Maria Abreu Consultant of: Prometheus Bioscience, Takeda, Pfizer, Janssen, Focus Medical Communications, Boehringer Ingelheim, Gilead, Imedex, Cornerstone Health, Landos Biophama, UCB, Eli Lilly, Bristol Myers Squibb, Arena Pharmaceuticals, and Cosmo Pharmaceuticals, Grant/research support from: Prometheus Bioscience, Takeda, Pfizer, Janssen, Focus Medical Communications, Boehringer Ingelheim, Gilead, Imedex, Cornerstone Health, Landos Biophama, UCB, Eli Lilly, Bristol Myers Squibb, Arena Pharmaceuticals, and Cosmo Pharmaceuticals, James Krueger Consultant of: AbbVie, Aclaris, Allergan, Almirall, Amgen, Arena, Aristea, Asana, Aurigene, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Escalier, Galapagos, Janssen, Eli Lilly, MoonLake Immunotherapeutics, Nimbus Lackshmi, Novartis, Pfizer, Sanofi, Sienna Biopharmaceuticals, Sun Pharma, Target-Derm, UCB, Valeant, and Ventyx, Grant/research support from: AbbVie, Akros, Allergan, Amgen, Avillion, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Exicure Incyte, Innovaderm, Janssen, Kyowa Kirin, Eli Lilly, Nimbus Lackshmi, Novan, Novartis, Parexel, Pfizer, Regeneron, UCB, and Vitae Pharmaceuticals, Kilian Eyerich Consultant of: AbbVie, Almirall, Boehringer Ingelheim, Bristol Myers Squibb, Hexal, LEO Pharma, Eli Lilly, Janssen, Pfizer, Novartis, Sanofi, and UCB, Kacey Sachen Shareholder of: Johnson & Johnson, Employee of: Janssen, Carrie Greving Shareholder of: Johnson & Johnson, Employee of: Janssen, Deepa Hammaker Shareholder of: Johnson & Johnson, Employee of: Janssen, Phuc Bao Shareholder of: Johnson & Johnson, Employee of: Janssen, Eilyn Lacy Shareholder of: Johnson & Johnson, Employee of: Janssen, Indra Sarabia Shareholder of: Johnson & Johnson, Employee of: Janssen, Janise Deming Shareholder of: Johnson & Johnson, Employee of: Janssen, Merle Elloso Shareholder of: Johnson & Johnson, Employee of: Janssen, Christopher T. Ritchlin Consultant of: UCB, AbbVie, Amgen, Lilly, Pfizer, Novartis, Gilead, and Janssen, Grant/research support from: UCB, AbbVie, Amgen, Lilly, Pfizer, Novartis, Gilead, and Janssen, Iain McInnes Shareholder of: Compugen, Evelo, and Causeway Therapeutics, Consultant of: Compugen, AstraZeneca, Bristol Myers Squibb, Amgen, Eli Lilly, GlaxoSmithKline, Janssen, Novartis, Roche, UCB, AbbVie, Cabaletta, Gilead, Pfizer, and Sanofi; Board member for: National Health Service Greater Glasgow and Clyde; Trustee of: Versus Arthritis, Grant/research support from: Compugen, AstraZeneca, Bristol Myers Squibb, Amgen, Eli Lilly, GlaxoSmithKline, Janssen, Novartis, Roche, UCB, AbbVie, Cabaletta, Gilead, Pfizer, and Sanofi, Matthieu Allez Speakers bureau: AbbVie, Amgen, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Janssen, Eli Lilly, Ferring, Galapagos, Gilead, IQVIA, Novartis, Pfizer, Genentech/Roche, Takeda, and Tillotts, Consultant of: AbbVie, Amgen, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Janssen, Eli Lilly, Ferring, Galapagos, Gilead, IQVIA, Novartis, Pfizer, Genentech/Roche, Takeda, and Tillotts, Grant/research support from: Janssen, Genentech/Roche, and Takeda, Anne Fourie Shareholder of: Johnson & Johnson, Employee of: Janssen.

Background: Differences in therapeutic profiles between IL-23 inhibitors have been observed across psoriatic disease domains, which may be related to their unique molecular attributes. Guselkumab is an IL-23p19-specific human IgG1 antibody, while risankizumab and tildrakizumab are humanized anti-IL- 23p19 IgG1 antibodies. Guselkumab and tildrakizumab have native Fc regions, while risankizumab has a mutated Fc region. We compared binding and functional characteristics of antigen-binding and Fc regions of these antibodies. Guselkumab displayed higher binding affinity for IL-23 (by surface plasmon resonance) than tildrakizumab but was comparable to risankizumab. Guselkumab also demonstrated higher potency (by inhibition of IL-23-induced STAT3 phosphorylation in human peripheral blood mononuclear cells) than tildrakizumab but was equivalent to risankizumab. In Fcy receptor (FcyR)- transfected cells, guselkumab and tildrakizumab showed strongest binding to CD64 (FcyRI); risankizumab had negligible binding to any FcyR. In interferon-y-primed human monocytes, guselkumab and tildrakizumab, but not risankizumab, showed dose-dependent binding to CD64 by flow cytometry. CD64- bound guselkumab and tildrakizumab were also able to bind IL-23. Guselkumab binding to CD64 on monocytes did not induce cytokine production. In conclusion, compared with risankizumab and tildrakizumab, guselkumab has unique attributes that confer the ability to neutralize IL-23 with high affinity and potency, as well as Fc binding to CD64+ myeloid cells. CD64+ mononuclear phagocytes are the dominant IL-23 source in psoriatic skin; therefore, guselkumab may be enriched within the inflamed tissue microenvironment by binding CD64 and poised to effectively neutralize IL-23 at its source, potentially leading to durable response and observed therapeutic differences within the class.

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFβ signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Clinically relevant differences between therapeutic antibodies against the same target may relate to their unique molecular attributes. Differences in therapeutic profile across the domains of psoriatic disease between guselkumab (GUS) and risankizumab (RIS) have been observed. To explore potential mechanisms underpinning this, we studied GUS, a fully human IgG1 specific for IL-23 with a native Fc region, and RIS, a humanized anti-IL-23 IgG1 with a mutated Fc region. We compared binding and functional characteristics of the antigen-binding and Fc regions of these antibodies. GUS and RIS displayed comparable picomolar affinities for binding IL-23 by KinExA and surface plasmon resonance assays, and equivalent potency (IC50 0.2 nM) for inhibition of IL-23-induced STAT3 phosphorylation in human peripheral blood mononuclear cells. However, in cells transfected with individual Fcγ receptors (FcγRs), GUS showed strongest binding to CD64 (FcγR1), while RIS showed negligible binding to any FcγRs, by virtue of its mutated Fc region. Furthermore, in IFNγ-primed human monocytes, labeled GUS showed dose-dependent binding to CD64 by flow cytometry, while RIS did not. GUS binding to CD64 on monocytes did not trigger activation as shown by lack of cytokine or chemokine production. Importantly, CD64-bound GUS was able to bind IL-23, as detected by anti-p40 staining. In conclusion, compared with RIS, GUS uniquely binds both CD64+ myeloid cells and IL-23. CD64+ mononuclear phagocytes are enriched in psoriatic skin and serve as the dominant IL-23 source. Taken together, GUS presence may be enriched within the inflamed tissue microenvironment by binding to CD64, neutralizing IL-23 at its cellular source, potentially leading to durable response and observed therapeutic differences within the class. Further studies are warranted to generate additional evidence supporting this hypothesis.

Clinically relevant differences amongst therapeutic antibodies against the same target may relate to their unique molecular attributes. Differences in therapeutic profile across the domains of psoriatic disease between guselkumab (GUS) and risankizumab (RIS) have been observed. To explore potential mechanisms underpinning this, we studied GUS, a fully human IgG1 specific for interleukin (IL)-23 with a native Fc region, and RIS, a humanized anti-IL-23 IgG1 with a mutated Fc region. We compared binding and functional characteristics of the antigen-binding and Fc regions of these antibodies. GUS and RIS displayed comparable picomolar affinities for binding IL-23 by KinExA and surface plasmon resonance assays, and equivalent potency (IC50=0.2 nM) for inhibition of IL-23-induced STAT-3 phosphorylation in human peripheral blood mononuclear cells. However, in cells transfected with individual Fcg receptors (FcgRs), GUS showed strongest binding to CD64 (FcgR1), while RIS showed negligible binding to any FcgRs, by virtue of its mutated Fc region. Furthermore, in interferon (IFN)g-primed human monocytes, labeled GUS showed dose-dependent binding to CD64 by flow cytometry, while RIS did not. GUS binding to CD64 on monocytes did not trigger activation as shown by lack of cytokine or chemokine production. Importantly, CD64-bound GUS was able to bind IL-23, as detected by anti-p40 staining. In conclusion, compared with RIS, GUS uniquely binds both CD64+ myeloid cells and IL-23. CD64+ mononuclear phagocytes are enriched in psoriatic skin and serve as the dominant IL-23 source. Taken together, GUS presence may be enriched within the inflamed tissue microenvironment by binding to CD64, neutralizing IL-23 at its cellular source, potentially leading to durable response and observed therapeutic differences within the class. Further studies are warranted to generate additional evidence supporting this hypothesis.

Objective Fibroblast‐like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase‐8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. Methods RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5‐8. Caspase‐8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase‐8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase‐8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase‐8B and G were determined. Results Caspase‐8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase‐8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase‐8) and induced by PDGF. The crystal structures of caspase‐8 variant G and B were identical except for a unique unstructured 59 amino acid N‐terminal domain in variant G. Selective knockdown of caspase‐8G was solely responsible for the effects of caspase‐8 on calpain activity and cell invasion in FLS. Conclusion Blocking caspase‐8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase‐8 inhibition.

Rheumatoid arthritis (RA) is a chronic autoimmune disease involving joint and bone damage that is mediated in part by proteases and cytokines produced by synovial macrophages and fibroblast-like synoviocytes (FLS). Although current biological therapeutic strategies for RA have been effective in many cases, new classes of therapeutics are needed. We investigated anti-inflammatory properties of the natural alkaloid tryptanthrin (TRYP) and its synthetic derivative tryptanthrin-6-oxime (TRYP-Ox). Both TRYP and TRYP-Ox inhibited matrix metalloproteinase (MMP)-3 gene expression in interleukin (IL)-1β-stimulated primary human FLS, as well as IL-1β-induced secretion of MMP-1/3 by FLS and synovial SW982 cells and IL-6 by FLS, SW982 cells, human umbilical vein endothelial cells (HUVECs), and monocytic THP-1 cells, although TRYP-Ox was generally more effective and had no cytotoxicity in vitro. Evaluation of the therapeutic potential of TRYP and TRYP-Ox in vivo in murine arthritis models showed that both compounds significantly attenuated the development of collagen-induced arthritis (CIA) and collagen-antibody-induced arthritis (CAIA), with comparable efficacy. Collagen II (CII)-specific antibody levels were similarly reduced in TRYP- and TRYP-Ox-treated CIA mice. TRYP and TRYP-Ox also suppressed proinflammatory cytokine production by lymph node cells from CIA mice, with TRYP-Ox being more effective in inhibiting IL-17A, granulocyte-macrophage colony-stimulating factor (GM-CSF), and receptor activator of nuclear factor-κB ligand (RANKL). Thus, even though TRYP-Ox generally had a better in vitro profile, possibly due to its ability to inhibit c-Jun N-terminal kinase (JNK), both TRYP and TRYP-Ox were equally effective in inhibiting the clinical symptoms and damage associated with RA. Overall, TRYP and/or TRYP-Ox may represent potential new directions for the pursuit of novel treatments for RA.

Background: The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing RA because of serum anti-citrullinated protein antibody positivity in absence of arthritis, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is through alterations in epigenetics patterns in adaptive immune cells. Objectives: Previous studies showed that DNA methylation patterns of early RA (ERA) synoviocytes differ from long-standing RA, suggesting that abnormal methylation occurs early in synovium and evolves over time. To extend these observations, we performed a cross-sectional analysis in TIP-RA of DNA methylation signatures in peripheral blood cells in ERA, at-risk anti-CCP3+ individuals and demographically matched CCP- controls. Methods: Genomic DNA was isolated from two independent cohorts of CCP- (cohorts 1 and 2, respectively: B cell: n = 17/34; memory T cell: n = 21/34; and naïve T cell: n = 21/33), CCP3+ (B cell: n = 18/37; memory T cell: n = 20/36; and naïve T cell: n = 20/35), and CCP3+ ERA (B cell: n = 4/18; memory T cell: n = 5/18; and naïve T cell: n = 5/18) after separating PBMCs using antibodies and magnetic beads. Methylation was measured by Illumina Infinium MethylationEPIC chip. Differentially methylated loci (DMLs) were identified using Welch’s t-test and mapped to gene promoter regions to define DM genes (DMGs). Principal component analysis (PCA) was used to represent relationship among groups. Pathway analysis was applied by Reactome. Results: For the initial cohort, 1494, 1097 and 1330 DMLs were identified among CCP+, CCP- and ERA in B cells, memory T cells and naïve T cells, respectively. For the confirmatory cohort, 523, 793 and 548 DMLs were found in corresponding cell populations. The DML overlap between the 2 cohorts was highly significant ( p = 2.48E-77). The DMLs were combined for both groups and corresponded to 411, 412, and 351 DMGs in B cells, memory T cells and naïve T cells. Of these, we found 246, 198 and 195 DMGs between CCP3+ and ERA in each peripheral blood cell population, respectively. PCA showed separation of CCP+, CCP- and ERA in each of the three blood cell types by DMLs (Fig. 1). DMGs were mapped to biological pathways to identify DM pathways. Although most were not significant, there were several highly significant differences comparing CCP+, ERA and CCP- in memory T cells involving pathways, including “Interferon gamma signaling” (FDR 7.48E-14), “PD-1 signaling” (FDR 8.71E-10), “Translocation of ZAP-70 to Immunological synapse” (FDR 4.75E-10), and “Phosphorylation of CD3 and TCR zeta chains” (FDR 8.71E-10). Figure 1. PCA shows the separation of CCP+, CCP- and ERA patients in memory T cells in confirmatory cohort. Conclusion: We identified reproducible methylation signatures of CCP-, CCP+, and ERA in peripheral blood B cells, memory T cells and naïve T cells in initial and confirmatory cohorts. The methylome of ERA also demonstrated a distinctive pattern from CCP+, indicating that progression to RA is accompanied by epigenetic remodeling, especially in T cell signaling and interferon responses. These signatures identify critical pathways in CCP positivity and classifiable RA and could provide the basis of novel interventions to prevent disease. Disclosure of Interests: Rizi Ai: None declared, David Boyle: None declared, Deepa Hammaker: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS, Andre Matti: None declared, William Robinson: None declared, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, Alyssa Johnsen Employee of: Janssen, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Wei Wang: None declared, Gary Firestein Grant/research support from: Lilly, Janssen, Abbvie

ObjectiveRheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) derived from hip and knee have distinctive DNA methylation and transcriptome patterns in interleukin (IL)‐6 signaling and Janus kinase (JAK)–signal transducers and activators of transcription (STAT) pathways. To determine the functional effects of these joint‐specific signatures, we evaluated how RA hip and knee FLS differ in their response to IL‐6.MethodsHip or knee RA FLS were obtained after arthroplasty. Previously published datasets on epigenetic landscape of FLS were mined to identify joint‐specific IL‐6–related epigenomic differences. RNA sequencing was performed on five RA hip and five knee FLS treated with or without IL‐6. Differential gene expression was determined using edgeR software. STAT3 phosphorylation was measured using bead assays. Sensitivity to tofacitinib was evaluated by measuring CCL2 inhibition using quantitative polymerase chain reaction.ResultsAssay for Transposase‐Accessible Chromatin sequencing and histone chromatin immunoprecipitation sequencing datasets from RA FLS were analyzed to identify epigenomic differences between hip and knee. Differential chromatin accessibility was associated with IL‐6, IL‐6R, and JAK1 genes. H3K27ac was also differentially marked at other JAK‐STAT–related genes, including STAT3‐STAT5A region. Principal component analysis of RNA sequencing data confirmed segregation between RA hip and knee FLS under basal conditions, that persisted following IL‐6 treatment. STAT3 phosphorylation after IL‐6 was significantly higher in knee than hip FLS and was highly correlated with JAK1 protein levels. Knee FLS were less sensitive to the JAK inhibitor tofacitinib than hip FLS.ConclusionRA hip and knee FLS have distinct transcriptomes, epigenetic marks, and STAT3 activation patterns in the IL‐6 pathway. These joint‐specific differences might contribute to a differential clinical response in individual joints to targeted therapies such as JAK inhibitors.